Globally, adolescent females are at highest risk for sexual HIV transmission, but yet also at risk for unwanted pregnancies. Sub-Saharan African adolescents utilize a range of contraceptives to prevent pregnancy. However, hormonal contraceptives have been implicated in increasing HIV risk in women. Contraceptives could influence HIV transmission by mediating effects on the levels and/or susceptibility of HIV target cells in the genital mucosa. Contraceptives might mechanistically mediate these HIV target cell changes by impacting the bacterial ecosystem (microbiome) at the vaginal mucosa. This study will be the first to evaluate the effects of contraception on two aspects of South African adolescent female genital tract that may render them at increased risk of HIV: genital HIV target cell susceptibility, and the genital microbiome. There are currently multiple available options for effective, reversible contraception in the public sector of South Africa (SA), and three of these will be assessed in this proposal. Norethisterone oenanthate (Net-En) is a long-acting injectable progestin used commonly in sub Saharan Africa. The other contraceptives contain both estrogen and progestin, and include combination oral contraceptives (COCPs) which need to be taken daily, and the Combined Contraceptive Vaginal Ring (CCVR), which is inserted into the vagina every 28 days. There is some evidence that the estrogen component may provide a protective benefit for HIV acquisition. This study will evaluate the hypothesis that in SA adolescent women, progestin-only contraceptives result in a skewed vaginal microbiome that leads to an increase in vaginal HIV target cell levels and susceptibility, compared to combination contraceptives. To address our hypotheses we will enroll 150 HIV-negative SA adolescents between the ages of 16 and 17 years from a separate randomized controlled trial of Net-En, COCPs, and CCVRs. Samples will be collected and evaluated at baseline for genital tract protein and gene expression, the phenotype/activation state of T cells from cervical cytobrush samples (by flow cytometry), and the genital microbiome (Aim 1). The young women will then be randomized to a contraceptive method, and will be followed longitudinally for 8 months. After 4 months, all participants will have their contraceptive method randomly switched. Aim 2 will evaluate the effect of contraceptives on the genital tract protein and gene expression, as well as evaluate the phenotype/activation state of T cells. Aim 3 will evaluate the vaginal microbiome during contraceptive usage utilizing 16S and metagenomic deep sequencing, and evaluate the findings from these aims in a cervical explant culture model. The studies outlined here may uncover why African adolescents are so disproportionately affected by HIV and has the potential to dramatically impact how contraception is administered to adolescents in developing countries to reduce HIV acquisition while allowing effective and safe family planning.